The Effection Of Receptor For Advanced Glycation End Products And Its Ligand HMGB1 In Rat Of Severe Acute Pancreatitis

Acute pancreatitis (AP), a common cause of acute abdominal pain, is usually a mild, self-limited disease. However, some 20% to 30% of patients develop severe disease manifested by pancreatic necrosis, abscess or pseudocysts, and/or extra-pancreatic complications such as vital organ dysfunction. Severe acute pancreatitis (SAP) is characterized by the development of systemic inflammatory respond syndrome and multiple organ dysfunction syndrome (MODS), as well as local pancreatic complications, and is still associated with a mortality rate of 15% to 30%, despite continuing improvement in critical care. Multiple organ dysfunction syndrome in the early phase and complications of infection in the late phase are contribution to high mortality in SAP. Multiple organ dysfunction syndrome is a consequence of the systemic inflammatory response syndrome, and it is conceivable that release of humoral mediators from the excessively activated macrophages, monocytes, and neutrophils may lead to the remote organ injury. Inflammatory reaction mechanism of the in-depth knowledge and research of severe acute pancreatitis, and the exploration on its treatment has always been hotspot.Currently, serum amylase and c-reactive protein has already been used to predict infection and prognosis in clinic, etc, but the application value is very limited in SAP.The Receptor for Advanced Glycation End Products (RAGE) first found on vascular smooth muscle cells, is a multiligand member of the immunoglobulin superfamily. RAGE is expressed in a variety of cell types, including macrophages, endothelial cells, vascular smooth muscle cells, mesangial cells, tumor cells, astrocytes, T lymphocytes. RAGE has been implicated in mediating the cytokine activity of HMGB 1. Its ligands include advanced glycation end products, amyloid peptide, S100 proteins as well as HMGB 1. RAGE-ligand interaction results in rapid and sustained cellular activation and gene transcription, diverse intracellular signals activated such as ERK1/2, (p44/p42), p38, SAPK/JNK, MAP kinases, rho-GTPases, phosphoinositol-3-kinase, and the JAK/STAT, in most cases culminating in the activation of nuclear factor-kappaB(NF-KB). This cellular activation is related to amplified and sustained inflammatory processes or tissue injury. RAGE knockout mice were recently reported to be resistant to septic shock induced by cecal ligation and puncture, suggesting that RAGE potentially plays a role in systemic acute inflammation.RAGE belongs to single span diaphragm section receptor, comprising all extracellular domain consisting of a single V-type immunoglobulin domain and two C-type immunoglobulin domains. Molecular study identifed that the ligand binding site existed within the V-domain by performing binding assays with truncations of the extracellular domain. After the extracellular domain, it follows a single transmembrane domain and a short 43 amino acid cytosolic tail. Recently, several carboxyl-terminal truncated isoforms of RAGE such as soluble RAGE(sRAGE)and endogenous secretory RAGE (esRAGE) were identified in the lung of both humans and mice. Because these isoforms lack a transmembrane domain, they were secreted and acted as decoy receptors and blocking RAGE to cell signal transduction. A soluble form of RAGE(sRAGE) is present in the circulation, comprises the extracellular domain of RAGE, and preserves its original ligand-binding capacity. The circulating pool of sRAGE consists of a splice variant named endogenous secretory RAGE (esRAGE) and proteolytically cleaved forms of RAGE. Elevated levels of sRAGE have been found in disorders such as end-stage renal disease, and acute lung injury, and reduced levels in rheumatoid arthritis, Alzheimer disease, and essential hypertension. At present, there are severe acute pancreatitis studies suggest merger organ failure, whether sRAGE are significantly different.High mobility group 1 protein (HMGB1), is one of the main ligands of the RAGE, named for its rapid migration operties on electrophoretic gels, also called amphoterin, is a member of the nonhistone romatin-associated proteins, and represents a novel group of intracellular proteins at function as inflammatory cytokines when released into the extracellular milieu. HMGB1 has three structure domain, which is A, B and C area, A and B area of main component are HMGB1 nonspecific DNA-binding area. C area and other protein can be interaction to adjust the affinity of HMGB1 and DNA. HMGB1 distribution is very extensive and expression in a variety of organic cells (such as lymphatic tissues, brain, liver, lungs, heart, spleen and kidney, etc.). It through two different ways release to extracellular, activation inflammatory cells actively secreted and the necrotic cells passively released. The recent study reports that HMGB1 seems to act as a late mediator involved in pathophysiology of sepsis, and the process of SAP from systemic inflammatory response syndrome (SIRS), to multiple organ dysfunction syndrome(MODS) and multiple organ failure (MOF), has also played a very important role. And with other promote inflammation factors such as tumor necrosis factor-a (TNF-a) and interleukin-1 (IL-1) different, HMGB1 has appeared late, but duration long characteristics, in order to expand the clinical treatment time window. Currently, the reach of SAP about RAGE and ligand HMGB1 is less, in order to determine the role of RAGE and ligand HMGB1 in SAP, we establish SAP rat model, to observe the dynamic changes of RAGE and ligand HMGB1 transcription and protein level in SAP pancreatic tissue after SAP mold is successful made, and to detect the serum level of sRAGE and HMGB1. This study is not only to provide a new thinking for clinical treatment SAP, but also to provide reference of inflammation mechanism involved in acute injury disease.Methods and results:1.The establishing of SAP model in ratsObjectives:To establish the animal model of SAP in rats and microscopically for the observation of pathological changes of the abdominal viscera.Methods:64 male Spraque-Dawley(SD) rats were randomly divided into 8 groups, with 8 rats in each group. The rat model of SAP was established by injecting intraperitoneally 20% L-arginine in at the two doses of 250mg/100g each, twice, 1h apart. Rats in control group were injected intraperitoneally with the same amount of saline. Rats were sacrificed at 6h,18h,24h,36h,48h,72h and 96h after SAP induction, heart blood collected, centrifugal separation serum after room temperature quiet place, kept at low temperature refrigerator after repacking serum. Meanwhile, the pancreas, liver, lungs and intestines samples were preserved. Determination of serum amylase(AMY), the pancreas and lungs samples were histologically examed by light microscope.Results:Serum amylase level began to increase at 6h after L-arginine intraperitoneal injection and reached a peak value at 18h, compared with controls. Ascites increased gradually at different time points in SAP rats, and reached the highest level at 96h. Microscopically,in control group, pancreatic acinar structure was complete, interlobular septa was clear, and there was mild edema but no inflammatory cell infiltration hemorrhage or necrosis in the pancreas. The pancreatic pathological score significantly increased at 6h after L-arginine intraperitoneal injection, in SAP 6h group, pancreatic tissue appeared edema, light or moderate inflammatory infiltration, but there was no damage of acinar structures. In SAP 18h group, pancreatic interstitial tissue appeared to be edema, mild necrosis and bleeding. In SAP 24h group, there were swelling pancreatic acinar cells, visible focal acinar cell necrosis, and inflammatory cells infiltration. And pancreatic necrosis and bleeding was more severe than that of SAP 18h group. Acinar structures destroyed more severely, and leaflets damage was more severe in SAP 36h group comparing with 24h group, and there were lots of inflammatory cell infiltration and acinar cells necrosis. In SAP 48h group. pancreatic tissue damaged seriously, hyperemia appeared surrounding the adipose tissue and inflammatory cells infiltration. In SAP 72h group, necrosis aggravated, acinar structures disappeared, nuclei dissolved, leaf gap widened, interstitial microvessel ruptured, lots of red blood cells overflowed, flake fat necrosis formated. In SAP 96h group, pancreatic tissue necrosis was at the highest level. Microscopically, in control group, pulmonary alveoli walls were complete, there was no inflammatory cell infiltration in interstitial lung. In SAP 24h group, interstitial lung widened, pulmonary alveoli cavity appeared pink drainage, accompanied by pulmonary alveoli atrophy and numerous neutrophils infiltrating. In SAP36h group, pulmonary alveoli interstitial widened obviously, pulmonary alveoli cavity significantly reddened, and there were lots of inflammatory cells infiltration. In SAP48h, SAP72h, SAP96h group, pulmonary alveoli structure disorded, pulmonary alveoli wall ruptured, the alveolar cavity bleeded obviously, the pulmonary alveoli interval widened significantly, and there were neutrophils infiltrating, structural damage in most of the pulmonary alveoli.Conclusions:The construction of SAP model in rat by intraperitoneal injection 20% L-arginine was simple, quick, repeatable, and stable.2. The expression of RAGE in rats with SAPObjectives:To detect the expression of RAGE in pancreas and the serum concentration of sRAGE in rats with severe acute pancreatitis.Methods:.Rats were sacrificed at 6h,18h,24h,36h,48h,72h and 96h after SAP induction. Pancreatic tissue was removed under aseptic technique. Total mRNA was extracted and reverse transcription-polymerase chain reaction (RT-PCR) amplification of a rat RAGE, amplified products gene in line with expectations. Real-time RT-PCR was performed to detect the gene expression of RAGE. Immunohistochemisty was performend to detect the protein expression of RAGE. Enzyme-linked immunosorbent adsorption method (ELISA) was used to detect serum sRAGE level.Results:The results of Real-time RT-PCR showed that gene expression of RAGE changed with the progression of the SAP. Statistics showed that the gene expression of RAGE in SAP 6h group, SAP 18h group, SAP 24h group, SAP 36h group, SAP 48h group, SAP7 2h group and SAP 96h group were significantly higher than that of control group so showed that the relative content gene expression of RAGE in SAP 24h group and SAP 36h group were significantly higher than that of other groups (P<0.05). The immunohistochemistry (IHC) confirmed that the RAGE protein was expressed in inflammation cells member and cytoplasm in SAP 48h group, and control group was no RAGE protein expression. It is also showed that the serum concentration of sRAGE were higher than that of controls in every time point(P<0.05). The serum concentration of sRAGE in SAP 18h group increased significantly, sRAGE concentration reached a peak value in SAP 24h group, compared with that of other groups (P<0.05). After 24h, serum concentration began to decrease, but sRAGE concentration remained at relatively higher than that of controls in SAP 36h groups and SAP 48h groups(T<0.05).Conclusions:RAGE plays an important role in the development of SAP. The expression level of RAGE in pancreatic tissues and serum are time dependence, and the serum level of RAGE may partly reflect the course of SAP.3.The expression of HMGB in rats with SAPObjectives:To detect the expression of HMGB1 in rats with severe acute pancreatitis.Method:Rats were sacrificed at 6h,18h,24h,36h,48h,72h and 96h after SAP induction. Pancreatic tissue was removed under aseptic technique. Extracted total mRNA from pancreatic tissure under aseptic technique, RT-PCR amplification of a rat HMGB1, amplified products gene in line with expectations. Real-time RT-PCR was performed to detect the gene expression of HMGB1. IHC was performend to detect the protein expression of HMGB1. ELIS A detection serum HMGB1 level.Results:Real-time RT-PCR results showed that gnen expression of HMGB1 chagned with the progression of the SAP. Statistics showed that gene expression of HMGB1 in SAP 6h group, SAP 18h group, SAP 24h group, SAP 36h group, SAP 48h group, SAP 72h group and SAP 96h group were significantly higher than that of control group (P<0.05). The relative content gene expression of HMGB1 in SAP 36h group were significantly higher than that of other groups (P<0.05). IHC showed that:in SAP 36h group, HMGB 1 expressed in nucleus and cytoplasm of pancreatic acinar cells and inflammatory cells and also found in pancreatic tissue mesenchyma. ELIS A results showed that the concentration of HMGB 1 in control group was lower than that of other groups(P<0.05). The serum concentration of HMGB1 reached a peak value in SAP 36h group, compared with other groups (P<0.05).Conclusions:HMGB1 seems to act as a late mediator in SAP, and appears in serum is later than its receptor RAGE.This study came to the following conclusions:1. The SAP rat model was established by injecting intraperitoneally of 20% L-arginine.2. RAGE plays an important role in the development of SAP. The expression level of RAGE in pancreatic tissues and serum are time dependence, and the serum level of RAGE may partly reflect the course of SAP.3. HMGB 1 seems to act as a late mediator in SAP.and appears in serum is later than its receptor RAGE.